KR101549457B1 - A novel conjugate of granulocyte colony-stimulating factor(g-csf) with polyethylene glycol - Google Patents

Abstract

The present invention relates to a novel physiologically active conjugate of drugs and medicines, that is, a granulocyte colony stimulating factor (G-CSF) represented by the formula (I) and relates to a conjugate having the activity of G-CSF. The present invention also relates to the use of a medicament containing the claimed conjugate represented by the formula (I), a pharmaceutical composition, a drug having a granulocyte colony stimulating factor as an active ingredient and a conjugate represented by the formula (I) An approach for preventing and / or treating neutrophils, a container containing a pharmaceutical composition:
(I)

Description

A novel conjugate of polyethylene glycol and granulocyte colony stimulating factor (G-CSF) {A NOVEL CONJUGATE OF GRANULOCYTE COLONY-STIMULATING FACTOR (G-CSF) WITH POLYETHYLENE GLYCOL}

The present invention relates to novel physiologically active conjugates of medicaments and medicaments, namely granulocyte colony stimulating factor (G-CSF), more specifically medicaments such as leukopenia, patients who have been treated with mainly bone marrow depression chemotherapy, hematopoietic stem cells To a novel conjugate of polyethylene glycol and G-CSF suitable for treating various types of neutropenia in transplant patients, chronic neutropenic patients, AIDS patients and other infected patients.

G-CSF is a hematopoietic growth factor that stimulates proliferation, differentiation and maturation of granulocytes (Metcalf, 1992). The introduction of exogenous G-CSF leads to rapid, specific and dose-dependent increases in neutrophils in peripheral blood (Welte et al., 1985; Hartung et al., 1995).

The human G-CSF gene was cloned and expressed in bacterial and mammalian cells; Various recombinant variants of human G-CSF (rhG-CSF) have been developed and refined (Nagata et al., 1986; Souza et al., 1986; Komatsu et al., 1987). this. The two rhG-CSF variants obtained in E. coli and CHO cells, respectively, are filgrastim and lenograstim. The Peel Grass team consists of 175 amino acids, is not glycosylated, and the amino acid sequence is the same as the native protein, but contains an additional methionine residue at the N-terminus of the molecule. The Renoglass team consists of 174 amino acids, is glycosylated, and the amino acid sequence fully matches the native human G-CSF sequence.

Various rhG-CSF products with different glycosylation and / or protein sequences may also be used to treat neutropenia, idiopathic, congenital and periodontal neutropenia occurring after chemotherapy; It is available for clinical use in combination with antibiotics to treat severe infections and to treat severe infections in neutrophil AIDS patients (Molineux, 2004).

However, clinical use of rhG-CSF was limited due to rapid absorption from subcutaneous injection sites, proteolytic instability, mass distribution, rapid clearance and short serum half-life (Mashkovsky, 2006; de Wit et al., 1996).

As a result, rhG-CSF must be administered to the patient on a daily basis for appropriate efficiency. The total number of peripheral leukocyte cells should be monitored daily.

The therapeutic efficacy of G-CSF can be improved when used in the form of a longer acting drug, wherein the natural protein molecule is chemically conjugated to monomethoxy polyethylene glycol (mPEG). PEGylation of G-CSF results in improved pharmacokinetics, increased half-life, reduced clearance, reduced concentration fluctuations in blood, decreased immunogenicity and toxicity, increased in vivo activity and increased stability (Molineux, 2003; Molineux, 2004) .

The biological properties of the PEG-G-CSF conjugate are mainly dependent on the molecular weight and the type of activated mPEG used. The reactive functional groups of the activated PEG can then be attached to specific portions of the protein, primarily amines, sulfhydryls or other nucleophilic groups. In most cases, the preferred site of modification is the N-terminus of the amino group and polypeptide chains of lysine (Kinstler et al. 1996, Roberts et al., 2002). A wide variety of mPEG derivatives have been used for amine PEGylation; For example, PEG-triazine, PEG-succinimidyl carbonate, PEG-succinimidyl succinate, PEG tresylate, PEG-aldehyde, PEG-N-hydroxysuccinimide-activated ester.

Several PEG-G-CSF conjugates are known.

Patent US 2007/0014762 discloses a PEG-G-CSF conjugate using various activated mPEGs having a molecular weight of 5000 Da, specifically mPEG-succinimidyl butanoate, mPEG-succinimidyl propionate, mPEG-succinimidyl a ≪ / RTI > methylbutanoate. To obtain the conjugate, rhG-CSF derived from CHO cells containing different amino acid substitutions at different positions was used. The bonding reaction was carried out at pH 7 to 9. The resulting PEG-G-CSF conjugate was composed of several positional isomers; Each isomer is conjugated to PEG 5000 at other sites with an epsilon-amino group of the lysine residue and an alpha-amino group of the N-terminal amino acid. The PEG-G-CSF conjugate was purified by ion exchange chromatography on an SP-Sepharose column.

There is no data on the activity and purity of the conjugates obtained in this patent.

Disadvantages of this final assembly are as follows:

1. Low molecular weight of mPEG conjugated to G-CSF;

2. Formation of several regioisomers in which PEG is conjugated to G-CSF through the free amino groups of several lysine residues and the N-terminal amino acid.

EP Patent 0401384 discloses a PEG-G-CSF conjugate (succinimidyl succinate, chloro-s-triazine, and the like) obtained using mPEG of a linear and branched structure with a molecular weight of 4,500 to 10,000 Da activated by other reactive groups. Polyoxyethylene diamine). The PEG-G-CSF conjugate showed a sustained effect in vivo, and the degree of persistence was dependent on the molecular weight of the conjugated PEG. The half-lives of PEG-G-CSF conjugate of PEG 10000 and unmodified G-CSF were 7.05h and 1.79h, respectively.

Disadvantages of the final conjugate are as follows:

The succinimidyl succinate-PEG was prepared by reacting mPEG with succinic anhydride and then activating the carboxylic acid with succinimidyl ester. The polymer backbone contains the remaining second ester linkage after the conjugation reaction with the protein. This bond is highly susceptible to hydrolysis after the polymer is attached to the protein. This hydrolysis not only loses the advantage of PEG attachment but also allows the succinate tag remaining in the hydrolyzed protein to act as a hapten and induce immunogenicity of the remaining protein (Roberts, 2002).

1. Chloro-s-triazine mPEG derivatives can be conjugated with functional groups of lysine and cysteine residues, resulting in the generation of many isomers, some of which are characterized by unstable linkages. Moreover, triazine mPEG derivatives are currently not used due to their high toxicity (Veronese & Pasut, 2005).

EP Patent 0335423 describes a conjugate of a triazine mPEG derivative having a molecular weight of 300 to 30,000 Da and G-CSF. This PEG-G-CSF conjugate exhibits a continuous action, and its activity is in the range of 11 to 60% of the unmodified G-CSF activity.

Disadvantages of the final conjugate are as follows:

1. Low inactivity.

2. Use of mPEG-triazine derivatives resulting in multiple positional isomers, some of which are characterized by unstable binding. Moreover, the mPEG-triazine derivatives are currently not used due to their high toxicity (Veronese & Pasut, 2005).

As a prototype of the present invention, the PEG-G-CSF conjugate described in US Pat. No. 5,824,784 (Example 2) was selected. This patent describes the reaction of reductive alkylation of activated colloidal rhG-CSF (ALD-mPEG) with activated propionaldehyde groups and linear mPEG with molecular weights between 6,000 and 25,000. The PEGylation reaction was carried out at pH 5. Under acidic conditions, the aldehyde is predominantly selective for the N-terminal a-amine due to the low pK of the a-amine relative to other nucleophiles. Coupling of the aldehyde proceeds through a Schiff base which is reduced in situ to provide a stable secondary amine linkage (Kinstler et al., 1996).

N-terminal PEGylation of rhG-CSF produced in this manner with PEG 20 kDa was used to develop the peg filler glass team ("Neulasta ") used for the treatment and prevention of multiple neutropenia conditions.

The mono-mPEG-G-CSF conjugate was separated by ion exchange chromatography using SP Sepharose HP column. The biological activity of the resulting purified PEG-G-CSF conjugate was 68% of native G-CSF activity. The content of unmodified G-CSF present in the final conjugate was 5% or less. The pharmacokinetic parameters of this conjugate were superior to unmodified G-CSF. Animal studies have shown that the introduction of the PEG-G-CSF conjugate increases the number of white blood cells and that the PEG-G-CSF conjugate has a more lasting effect than the unmodified G-CSF. When using a single dose of the G-CSF PEG-conjugate, the leukocyte cell content of the animals reached a maximum after 1 day of injection, this level remained during that day, and the leukocyte cell level decreased to baseline levels 4 days after injection . Upon introduction of unmodified G-CSF, the maximum level of leukocytes present in animal blood was observed 1 day after injection, and the leukocyte content rapidly decreased.

Disadvantages of the final conjugate are as follows:

1. Low inactivity (68% of unmodified G-CSF activity) of the resulting conjugate;

2. Presence of unmodified G-CSF in the conjugate.

It is an object of the present invention to provide a stable, highly refined novel < RTI ID = 0.0 > novel < / RTI > PEGylated G-CSF conjugate, as well as a pharmaceutical composition based on this conjugate.

This problem is solved by the preparation of a novel functional active molecule of PEG-G-CSF conjugate with activity of G-CSF, wherein the linear PEG molecule with a molecular weight of 30000 Da is strictly an alpha amino acid of N-terminal amino acid (methionine) To form a compound represented by the following formula (I): < EMI ID =

(I)

Wherein n is an integer from 681 to 1000; m is an integer of > = 4; N ^alpha HG-CSF is a natural or recombinant polypeptide having the activity of G-CSF.

In the conjugate thus derived, the linear PEG with a molecular weight of 30000 to 40000 Da is conjugated to the alpha amino group of the N-terminal amino acid of the G-CSF molecule.

The selectivity of the G-CSF modification via the alpha-amino N-terminal amino acid is achieved through the use of an aldehyde-activated mPEG represented by the following formula (II) and a PEGylation reaction at pH? 5:

(II)

Wherein n is an integer from 681 to 1000; The molecular weight of PEG is about 30000 to 40000 Da; m is an integer of? 4.

The optimal ratio of PEG molecular weight to specific G-CSF activity is achieved through the use of activated mPEG (II) with m > = 4, which results in more effective interaction of the receptor with the claimed conjugate herein, . The decreased immunogenicity, toxicity and improved pharmacokinetic parameters are achieved by increasing the molecular weight of the attached PEG. The novel features of the present invention as compared to the prototype are as follows:

1. Aldehyde derivatives of activated mPEG (Formula II) are used in the production of PEG-G-CSF claimed herein;

2. The formula of the PEG-G-CSF conjugate is novel;

3. The molecular weight of the PEG-IFN conjugate increases due to the molecular weight of the PEG;

4. The level of inactivity is higher;

5. The pharmacokinetic parameters are improved.

The PEG-G-CSF conjugates claimed herein are highly purified and have a lasting effect and are characterized by high biological inactivity (at least 84-94% of unmodified G-CSF), protein purity ≥97%, high thermal stability, proteolytic enzyme activity , Improved pharmacokinetic parameters. ≪ / RTI >

The present invention also encompasses a pharmaceutical composition containing the PEG-G-CSF conjugate claimed in the present invention in an amount effective as an active ingredient. The composition may also contain pharmaceutically acceptable carriers, buffers (organic and inorganic acids and salts thereof such as citrates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, acetate buffers), stabilizers (sugar alcohols, Amino acids, organic sugars or sugar alcohols, inositol, polyethylene glycol, polymers of amino acids, sulfur-reducing agents such as urea, glutathione and thioglycerol, low molecular weight proteins such as human serum albumin, polypeptides with immunoglobulins, polyvinylpyrrolidone Polysaccharides such as raffinose, and polysaccharides such as dextran), preservatives (such as phenols, benzyl alcohols, benzyl alcohols, benzyl alcohols, Methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium chloride, (Sodium chloride, sorbitol, mannitol, arabitol, xylitol, etc.), non-ionic surfactants, and surfactants (polysorbates, Such as a lyophilizate or a liquid formulation (such as an injection, a spray, a drop, etc.), and the like. Can be applied.

The invention also encompasses medicaments based on the claimed conjugates, particularly medicaments for the treatment of neutropenia.

The claimed PEGylated G-CSF, as well as the pharmaceutical compositions and medicinal products based thereon, are useful in the treatment of neutropenia occurring after chemotherapy, in the tolerance of immunosuppressive drugs at the time of bone marrow transplantation, in the immunization of AIDS and other infections Can be used to improve the condition of patients, especially patients with systemic or transmissible candidiasis.

The conjugate of formula (I) is administered intravenously, subcutaneously, intramuscularly, or by any other suitable technique with an optional pharmaceutically acceptable filler, diluent and / or pharmaceutically acceptable excipient. The route of administration will depend on, for example, symptoms and age, frequency of administration and interval of injection. Depending on the disease and its severity or purpose of administration (therapeutic or prophylactic), the effective amount of the PEG-G-CSF agent is selected according to the factors mentioned above.

In order to obtain the claimed conjugate PEG-G-CSF, a highly purified recombinant human G-CSF (Peachgrass team, CJSC product "Biocad") is a butyraldehyde mPEG derivative of the formula (II) with a molecular weight of 30000 to 40000 Da .

The bonding reaction was carried out at a temperature of 20 DEG C or lower and in the presence of a reducing agent at a pH of 5.0 or less. The molar ratio of PEG / protein was 2.5 to 5 / l. The reaction procedure was monitored by SDS-PAGE and RP-HPLC under reducing conditions. G-CSF (one PEG per molecule) by cation exchange chromatography from the reaction product (undesired form of PEG-G-CSF containing two or more chains of PEG per protein molecule and unmodified G-CSF) Gt; G-CSF < / RTI > bearing the chain) was purified. The mono PEG-IFN conjugate elution was carried out with a linear gradient of 0.05 to 0.2 M sodium chloride concentration in buffer solution below pH 5. The purified monoPEG-G-CSF is dialyzed against a 10 to 50 mM buffer solution at a pH of 4 to 5, and the salt, or polysaccharide, or alcohol, or polyvinylpyrrolidone, or monosaccharide and amino sugar, And a non-ionic surfactant were added, and the surface was stored in a silicone-treated plastic bottle or glass bottle at 4 ± 2 ° C.

In order to characterize the final mono PEG-G-CSF conjugate, purity, uniformity, physical-chemical, biological and pharmacokinetic parameters were compared to unmodified G-CSF.

The invention is illustrated by the following figures:

Figure 1 is the kinetics of G-CSF PEGylation.
Figure 2 is a reverse phase high performance liquid chromatogram (RP-HPLC) of a PEG-G-CSF conjugate.
Figure 3 is a high performance liquid chromatogram (SEC-HPLC) of size-exclusion of PEG-G-CSF conjugate.
Figure 4 shows the results of SDS-PAGE analysis of PEG-G-CSF conjugate (line 1) and unmodified G-CSF (line 2).
5 is a MALDI mass spectrum of unmodified G-CSF (A) and PEG-G-CSF conjugate (B).
Figure 6 compares the mass spectra of trypsin-degrading peptides of G-CSF (A) and PEG-G-CSF conjugate (B).
Figure 7 shows the thermal stability of the PEG-G-CSF conjugate (green line) and unmodified G-CSF (red line) at + (50 ± 2) ° C.
Fig. 8 shows the proteolytic stability of PEG-G-CSF conjugate (green line) and unmodified G-CSF (red line).
Figure 9 shows the immunoreactivity of the PEG-G-CSF conjugate (green line) and unmodified G-CSF (red line).
Figure 10 shows the pharmacokinetics of the PEG-G-CSF conjugate (green line) and unmodified G-CSF (red line).

An example and performance study of the specific performance of the PEG-G-CSF production process is presented below.

Example  One

The recombinant human G- CSF ( rhG - CSF , Phil Grass Team )

The separation and purification of rhG-CSF was performed as described in RU Patent 2278870.

Example  2

pegged rhG - CSF Manufacturing

50 ml of 1 M sodium cyanoborohydride solution was added to 2300 ml of buffer (50 mM sodium acetate, pH 5.0 + 0.2) containing 2500 mg of purified rhG-CSF prepared according to Example 1. After stirring the mixture, 10 g of solid mPEG-ButyrALD (methoxy-PEG-butyraldehyde) having an average molecular weight of 30 kDa was added. The junction reaction mixture was stirred at (20 ± 2) ° C for 22 hours. After various time intervals, 50 [mu] l samples were taken from the reaction mixture and the dynamics of PEG-G-CSF conjugate formation were analyzed using SDS-PAGE at 12.5% gel under reducing conditions. For this, 17 μl of a buffer containing 125 mM Tris-HCl, pH 6.8, 20% glycerol, 3% SDS, 15% 2-mercaptoethanol and 0.005% bromophenol blue was added to the collected samples. Min. A 5 [mu] l volume of sample was loaded into each well. SDS-PAGE was performed using a Mini Protean System (BioRad). The gel was stained with Coomassie R-250. The kinetics of G-CSF PEGylation are presented in FIG. At a time when the content of PEG-G-CSF exceeded 70%, the reaction mixture was diluted 3-fold in 10 mM sodium acetate buffer, pH 4.8 ± 0.2.

Example  3

Mono PEG -G- CSF  Conjugate purification

The diluted reaction mixture prepared according to Example 2 was loaded into a column filled with 300 ml of CM-Sepharose equilibrated with 10 mM sodium acetate buffer pH 4.8 (Buffer A) at a flow rate of 10 ml / min. After loading the mixture, the column was washed with 1500 ml of Buffer A to remove unbound material. Mono PEG-G-CSF was eluted from the column using a 0 to 0.2 M NaCl linear gradient in Buffer A (6000 ml) at a flow rate of 5 ml / min. Fractions (50 ml) were collected and absorbance was measured at 280 nm and 260 nm. Fractions containing protein were analyzed by RP-HPLC and SDS-PAGE as described in Example 2. [ Fractions containing monoPEGylated G-CSF in a purity greater than 95% were pooled and dialyzed against 10 volumes of 1.6 mM sodium acetate buffer, pH 4.0 +/- 0.2. Sterile filtration was then performed and the samples were stored at 4 + 2 [deg.] C.

Example  4

Inverse phase  High Performance Liquid Chromatography ( RP - HPLC )On by PEG -G- CSF  Purity analysis of conjugate

The purified PEG-G-CSF conjugate prepared according to Example 3 was diluted to a protein concentration of 0.3 mg / ml with 20 mM sodium acetate buffer, pH 5.0. Analytical RP HPLC was performed by UV detection at 214 nm using a gradient Waters 'Breeze' chromatograph on a C4 Symmetry column (4.6 cm x 150 mm). 100 쨉 l of the obtained sample was injected into the column. The mono PEG-G-CSF was eluted with a single symmetrical peak from the column by RP_HPLC (FIG. 2) and was found to be of high purity when judged by only a small amount of impurities eluting before and after the main UV absorption peak (FIG. 2) . The impurities were less than 1.15% of the material eluting from the main peak. Thus, it can be concluded from the presented data that the purity of the claimed PEG-G-CSF conjugate is ≥98% according to RP-HPLC.

Example  5

High Performance Size Exclusion Chromatography ( SEC - HPLC )On by PEG -G- CSF  Purity analysis of conjugate

The purified PEG-G-CSF conjugate prepared according to Example 3 was diluted to a protein concentration of 0.3 mg / ml with 20 mM sodium acetate buffer, pH 5.0. Analytical SEC HPLC was performed by UV detection at 214 nm using a Waters 'Breeze' chromatograph on a TSK G3000SWX column (30 cm x 7.8 mm). 100 쨉 l of the obtained sample was injected into the column. SEC-HPLC results are shown in Fig. The PEG-G-CSF sample eluted from the column as a unique symmetric peak was found to be 98.47%. From the results shown in Fig. 3, it can be seen that the claimed PEG-G-CSF conjugate does not contain free-modified G-CSF. The content of high molecular weight aggregates in the claimed conjugate does not exceed 1.53%. The purity of the claimed PEG-G-CSF conjugate according to SEC HPLC is greater than 98%.

Example  6

PEG -G- CSF  Measurement of endotoxin levels present in the conjugate

The content of bacterial endotoxin (BE) in the PEG-G-CSF conjugate sample prepared according to Example 3 was determined according to the requirements of FFS 42-0002-00 using the Lug method (a modification of the gel coagulation test) Lt; / RTI &gt; in vitro. The multi-component diagnostic kit << Associates of CAPE COD, Inc. >>, LAL-reagent PYROTELL® (sensitivity 0.03 EU / ml), Endotoxin Reference Standard (CSE, 0.5 mg Bayer) and LAL-test water were used. The results presented in Table 1 show that the BE content in the PEG-G-CSF conjugate sample is below 3EU per mg of protein (endotoxin unit), which is much lower than the BE levels allowed in drugs based on recombinant proteins.

Bacterial endotoxin content in PEG-G-CSF conjugate PEG-G-CSF conjugate Bacterial endotoxin content (EU / mg protein) PEG-G-CSF prepared according to Example 3 ? 3.0

Example  7

SDS - PAGE On by PEG -G- CSF  Purity analysis of conjugate

Samples of purified PEG-G-CSF conjugate prepared according to Example 3 were analyzed by SDS-PAGE as described in Example 2. A sample containing 40 쨉 g of both PEG-G-CSF and unmodified G-CSF was loaded into each well. The gel was stained with Coomassie R-250. Figure 4 shows the SDS-PAGE profile of PEG-G-CSF (line 1) and the SDS-PAGE profile of unmodified G-CSF (line 2). The purified PEG-G-CSF appeared as a single diffusion band with an apparent molecular weight higher than that of unmodified G-CSF. It should be noted that the exact molecular weight value of the PEGylated protein can not be determined by SDS-PAGE because the conjugation of the hydrophilic PEG molecule to the protein increases the Stokes radius of the PEGylated protein. As a result, the electrophoretic mobility of the PEG-protein complex in the gel is slowed down and its molecular weight value is much higher than the unmodified protein and PEG molecular weight sum.

Example  8

Unmodified G- CSF  prepare, PEG -G- CSF  Junction Mass spectrometric  Molecular weight measurement

The true molecular weight of PEG-G-CSF was measured using MALDI-TOF MS (Matrix assisted laser desorption time-of-fight mass spectrometry). MALDI TOF MS was performed with an Ultraflex II (Bruker Daltonics) mass spectrometer equipped with a UV laser (Nd). 1 μl of a PEG-G-CSF conjugate sample prepared according to Example 3 and 0.3 μl of a 2,5-dihydroxybenzoic acid solution (Aldrich, 10 mg x ml ^-1 in 20% acetonitrile aqueous solution containing 0.5% TFA) Mixed and dried in air. Unmodified G-CSF samples were prepared in the same manner.

The mass spectrum was obtained in the positive mode MS spectrum and the average mass error did not exceed 10 to 15 Da.

The MALDI-TOF mass spectrum of PEG-conjugated G-CSF and unmodified G-CSF in the range of m / z 10000 to 50000 is presented in FIG.

Figure 5A shows that the mass spectrum of G-CSF contains a main peak corresponding to a single chargeable ion [M] ^&lt; ⁺ & gt ^; having a molecular weight of 18,816 Da.

In the mass spectrum of the PEG-G-CSF conjugate shown in Figure 5B, there is a broad spectral peak with a molecular weight center around 49,600 Da. The broad spectrum is due to the heterogeneous population of PEG polymer species.

Given the molecular weight of G-CSF (19,295 Da) and PEG (30,000 Da), the molecular weight of the resulting conjugate (49,600) is very consistent with the calculated mass sum of each molecule.

Example  9

PEG -G- CSF  In the conjugate PEG Measurement of area

15 mcg x ml ^-1 concentration as was added to the modified trypsin (Promega) solution 5㎕ dissolved in 0.05M NH ₄ HCO ₃ in the PEG-G-CSF conjugate probe 5㎕ prepared according to Example 3. The hydrolysis was carried out at a temperature of 37 ° C for 16 hours, and then 10 μl of 0.5% TFA in a 10% aqueous solution of acetonitrile was added to the solution, followed by thorough mixing. Unmodified G-CSF samples were also prepared in the same manner. The tryptic digest was analyzed by MALDI-MS.

The site of PEG junction with the G-CSF molecule was determined by comparing the mass spectra of the trypsin-degrading peptide of the PEG-G-CSF conjugate and the unmodified G-CSF. During trypsin digestion of the PEG-G-CSF conjugate, there should be no peptides corresponding to the portion of the protein that has undergone strain. Table 2 presents the mass spectra of experimental trypsin-degrading peptides of unmodified G-CSF and PEG-G-CSF conjugates. This table shows that the mass spectrum of the trypsin-degrading peptide of unmodified G-CSF closely matches that of the PEG-G-CSF conjugate (Table 2). However, there is no peptide with a molecular weight of 1432.7 present in the tryptic digest of the unmodified G-CSF in the trypsin digest of the PEG-G-CSF conjugate. The mass spectra obtained experimentally from trypsin-degrading peptides of the unmodified G-CSF and PEG-G-CSF conjugates are shown in FIG. 6, which shows that peptides with a molecular weight of 1432.7 were added to the trypsin degradation product of the PEG- &Lt; / RTI &gt; According to theoretical mass spectrometry of trypsin-degrading peptides of G-CSF, the peak at m / z 1432.7 corresponds to the N-terminal peptide of the protein (Table 2). The absence of this peak in the tryptic lysate of the PEG-G-CSF conjugate is consistent with the modification of the N-terminal peptide, which became heavier by the weight of the PEG, deviating from the region of the investigated specimen. The conjugation of the G-CSF molecule with PEG occurred at the amino group of the N-terminal methionine, the only free amino group present in this peptide.

Experimental mass spectra of trypsin-degrading peptides of PEG-G-CSF conjugate and unmodified G-CSF compared to theoretical values M / z of the experimental peptide Theoretical peptide G-CSF PEG-G-CSF mass Peptides order 1432.7 - 1432.75 1-14 MTPLGPASSLPQSF 747.4 747.4 747.36 18-23 CLEQVR 1129.6 1129.6 1129.61 20-29 EQVRKIQGDG 1359.7 1359.7 1359.75 42-53 LCHPEELVLLGH 2527.3 2527.3 2527.30 54-78 SLGIPWAPLSSCPSQ 953.6 953.6 952.56 55-63 LGIPWAPLS 818.4 818.4 817.41 61-68 PLSSCPSQ 2033.1 2033.1 2033.10 72-90 LAGCLSQLHSGLFL 2104.1 2104.1 2104.14 75-93 CLSQLHSGLFLYQG 1241.7 1241.7 1241.68 158-167 LQSFLEVSYR

Example  10

Unmodified rhG - CSF  prepare, PEG -G- CSF  Measurement of inactivity of specific conjugates

A PEG-G-CSF conjugate prepared according to Example 3 and diluted with RPMI medium at a concentration of 0.2 ng / ml and serial dilutions of the sample to be tested were prepared in the assay medium. Serial dilutions of the international reference standards (1-st International Standard 88/502, granulocyte colony stimulating factor, human, rDNA-derived, 10000 IU / amp) of unmodified G-CSF and G-CSF activity were analyzed side by side. 100 占 퐇 of the diluted protein sample was added to the test wells of the flat 96-well tissue culture plate. For bioassay, M-NFS cells were washed and resuspended in RPMI medium at a concentration of 1.5x10 &lt; ⁵ &gt; / ml. 100 占 퐇 of cell suspension was added to each well and cultured in a 5% CO ₂ tissue culture incubator for 50 hours at 37 占 폚. Next, 20 [mu] l of the dye Alamar Blue (or its analogue, e.g. MTT) was added to each well and the plates were incubated for 14 hours under the same conditions.

The increase in cell count was measured using an optical flatness reader at an optical density at wavelengths of 545 nm and 630 nm. The specific biological activity (B ₀ ) (IU / mg) of the tested samples was calculated using the following equation:

B ₀ = A ₀ / C

Here, the biological inactivity (IU / mg) of B ₀ -PEG-G-CSF or G-CSF;

Biological activity of A ₀ -PEG-G-CSF or G-CSF (IU / mg);

Protein content (mg / ml) in C-PEG-G-CSF or G-CSF solution.

PEG-G-CSF and unmodified G-CSF Product Inactive (IU / mg) The PEG-G-CSF conjugate prepared according to Example 3 (0.845 + 0.091) x 10 &lt; ^{8 &gt;} Unmodified G-CSF (1.00 + 0.081) x 10 &lt; ^{8 &gt;}

Example  11

Unmodified G- CSF  prepare, PEG -G- CSF of Thermal stability  analysis

A PEG-G-CSF conjugate sample prepared according to Example 3 and diluted to a protein concentration of 0.28 mg / ml or less in 5 mM sodium acetate buffer pH 4.0 containing 140 mM NaCl was placed in a water bath at (50 ± 2) The turbidity of the protein solution was measured at various time intervals by measuring the absorbance at 340 nm. Similarly, the investigation of the thermal stability of the unmodified G-CSF was carried out. Incubation of the protein at (50 ± 2) ° C. forms an insoluble aggregate, which can increase the turbidity of the protein solution, which can be measured by optical density analysis of the solution at 340 nm. Figure 7 shows the results of this study, wherein the turbidity of the unmodified G-CSF preparation increased with incubation time, while the PEG-G-CSF product remained stable throughout the entire observation period. This data suggests that the thermal stability of the claimed PEG-G-CSF conjugate is much higher than that of unmodified G-CSF.

Example  12

Unmodified G- CSF  prepare, PEG -G- CSF Protein degradation stability

The PEG-G-CSF conjugate sample prepared according to Example 3 was diluted to a concentration of 0.6 mg / ml, and 150 μl of 1 M Tris-HCl (pH 8.5) was added to 850 μl of the final sample. Then, ₂ μl of 0.5 M CaCl ₂ was added to a final concentration of 1 mM, and after stirring, 15 μl of trypsin (Promega, Gold) was added to a final concentration of 0.02 μg / ml. Similarly, a sample containing unmodified G-CSF was also prepared. The samples were incubated at 37 &lt; 0 &gt; C. A sub-sample (100 μl) was taken every 60 minutes and 20 μl of 10% SDS was added to stop the trypsin reaction and SDS-PAGE was performed as described in Example 1. After electrophoresis, the gel was stained with Coomassie-R-250 and the concentration measurement of the stained gel was performed. The area of the main band was measured with a computer program GelPro Analyzer. The area of the main band of the initial sample (before trypsin treatment) was regarded as 100%. Figure 8 shows sensitivity data for trypsinization of the PEG-G-CSF conjugate and G-CSF. It is clear from this that as the trypsin treatment time increases, the PEG-G-CSF and G-CSF contents decrease, but the claimed PEG-G-CSF conjugate is more resistant to trypsin action than unmodified G-CSF .

Example  13

Unmodified G- CSF  prepare PEG -G- CSF  Immunoreactivity of the conjugate

Immunoreactivity of the claimed PEG-G-CSF conjugate was measured by antibody binding activity by modified ELISA assay using ProCon G-CSF ("Protein contour" LLC) reagent set. The PEG-G-CSF conjugate prepared according to Example 3 was diluted to 1 mkg / ml in 10 mM Tris-HCl buffer (pH 7.2) containing 140 mM NaCl and 1% bovine serum albumin. Next, 100 占 퐇 of the obtained sample was added to wells of a microtiter plate previously coated with a monoclonal antibody against G-CSF. After incubation at 37 占 (60 min), monoclonal antibodies to each epitope, labeled with biotin, were added to the wells. The resulting immunocomplexes were detected using streptavidin labeled with horseradish peroxidase. To induce a color reaction in the wells, 100 μl of 0.1% H ₂ O ₂ in a 0.05 M TB (pH 7.2) and a substrate solution (200 μg / ml tetramethylbenzidine (Sigma)) was added and the mixture was incubated did. This reaction was stopped by adding 50 μl of a 2 M sulfuric acid solution to the wells. The results were taken by measuring the absorbance at a wavelength of 405 nm in a microplate reader << Multiscan >> EX. Likewise, the immunoreactivity of unmodified G-CSF was also examined. The interaction of unmodified G-CSF with immunoglobulin was considered 100%. From the results shown in Fig. 9, it can be seen that the immunoreactivity of the claimed PEG-G-CSF conjugate is 25% of the immunoreactivity of unmodified G-CSF.

Example  14

During storage PEG -G- CSF  Measurement of the stability of the conjugate

Samples prepared according to Example 3 were diluted to 1 mg / ml in 1.6 mM sodium acetate buffer (pH 4.0 +/- 0.2). A small amount of the diluted sample was taken and placed in a sterile tube with a "Eppendorf" type lid. And stored at a temperature of (6 2) ° C. The inactivity and uniformity of the PEG-G-CSF conjugate were measured at various storage time intervals. The uniformity of PEG-G-CSF was analyzed by SDS-PAGE and SEC-HPLC as described in Example 1 and Example 5, respectively. Table 4 shows that the PEG-G-CSF conjugate is stable for at least 24 months when stored at temperature (6 +/- 2) &lt; 0 &gt; C.

Stability of the claimed PEG-G-CSF conjugate at (6 ± 2) parameter Shelf life (months) 0 6 12 18 24 Active (IU / mg) (0.84 0.09) x 10 ⁸ (0.75 + - 0.15) x10 &lt; ⁸ &gt; (0.9 +/- 0.10) * 10 &lt; ^{8 &gt;} (0.80 + 0.15) * 10 &lt; ⁸ &gt; (0.86 + 0.07) x10 &lt; ^{8 &gt;} Uniformity by HPLC GF (%) > 95 > 95 > 95 > 95 > 95 Uniformity (%) by EF in PAGE > 98 > 98 > 98 > 98 > 98

Example  15

PEG -G- CSF  Pharmacokinetic analysis of conjugates

A 5 샘플 sample of the PEG-IFN conjugate prepared according to Example 3 was injected intraperitoneally into an ICR-type male mouse (20 g). In parallel, unmodified G-CSF was injected into the other groups of mice. Serum samples were taken by puncture posterior to the eye at different times after injection. Blood serum was obtained by standard methods.

Serum samples to measure either native or monoPEGylated G-CSF were used for ELISA analysis using mouse monoclonal immunoglobulin against several epitopes of human G-CSF.

The wells of the microtiter plates were coated with monoclonal antibody to G-CSF (250 ng / well in 100 μl of 20 mM Tris-HCl buffer (TB) (pH 9.0)). After overnight incubation at 4 ° C, 200 μl of TB (pH 7.4) containing 1% bovine serum albumin (BSA) and 0.05% Tween-20 was added to the wells to block non-specific binding sites. Then 100 μl of the analyzed serum diluted with TB containing 1% BSA and 0.05% Tween-20 in a ratio of 1: 2, 1: 4 and 1: 8 was added to the wells. To construct a standard calibration curve in parallel, a PEG-G-CSF sample of known concentration range from 25 ng / ml to 600 ng / ml was added to the well in TB buffer containing 1% BSA and 0.05% Tween-20. After incubation at 37 占 폚 for 1 hour, the biotinylated anti-human G-CSF monoclonal antibody and the streptavidin-peroxidase conjugate were added successively to the wells. After incubation and washing to remove any unbound enzymes, 100 μl of substrate solution (100 mM sodium acetate buffer containing 0.015% hydrogen peroxide and 0.2 mg / ml tetramethylbenzidine, pH 5.0) was added to induce a color reaction product . The reaction was quenched by the addition of 2M H ₂ SO ₄ . The optical density was measured at 450 nm in a microplate reader. The concentration of G-CSF present in the serum was measured using a calibration curve and taking into account the dilution rate of the irradiated serum.

A calibration curve constructed with a known natural G-CSF concentration (ranging from 6.25 ng / ml to 200 ng / ml in buffer TB containing 1% BSA and 0.05% Tween-20) was used for unmodified G-CSF pharmacokinetic analysis Used.

The results of the claimed PEG-G-CSF pharmacokinetic are presented in FIG. 10 as compared to unmodified G-CSF. These results suggest that the claimed PEG-G-CSF conjugate has a significant sustained effect. Upon administration of unmodified G-CSF, the concentration of this G-CSF present in the animal blood reached a maximum at 1 h post-injection and then rapidly decreased. Upon administration of the PEG-IFN conjugate, the maximum amount of G-CSF in the animal blood was observed after 6 hours (Fig. 10), and then a slow decrease of G-CSF in the blood was observed for 140 hours. Based on this result (FIG. 10), the main pharmacokinetic parameters of the claimed PEG-G-CSF conjugate were calculated. The results show that the absorption, distribution volume and removal rate of the PEG-G-CSF conjugate from the injection site is much slower than that of the unmodified G-CSF resulting in a long-term circulation in the blood of the PEG-G- Time out).

Example  16

Charged PEG -G- CSF  Conjugate and round ( prototype ) Method compared to PEG-G-CSF conjugates

The structure, basic physico-chemical and pharmacokinetic parameters of the PEG-G-CSF conjugate as described in the circular method and the claimed PEG-G-CSF conjugate obtained according to Example 3 were compared (Table 5). The data presented in Table 5 show that the properties of the claimed PEG-G-CSF conjugate are significantly improved over the conjugates of the circular method.

The major physico-chemical and pharmacokinetic parameters of the claimed PEG-G-CSF conjugate versus the PEG-G-CSF conjugate as described in the circular method (US patent 5,824,784) parameter
PEG-G-CSF conjugate The claimed PEG-G-CSF conjugate The conjugates described in the circular method (US Pat. No. 5,824,784) G-CSF Phil Grass Team Phil Grass Team PEG structure Linear Linear PEG molecular weight (kDa) 30-40 6-25 PEG-G-CSF conjugate structure *

The inactive% of unmodified G-CSF 84-94 68 Purity by PAGE (%) 100 ** Purity results by HPCL (%) > 98 ** Purity results by GF HPLC (%) > 98 ** Bacterial endotoxin content (EU / mg) <3 ** Pharmacokinetic parameters The AUC ratio of PEG-G-CSF / G-CSF 18 **

* - PEG-G-CSF conjugate structure obtained by the circular method presented in WHO Drug Information, 2,002, v.16, N.1, 101,

** - no data.

Example  17

Subcutaneous injection solution 0.6 mg / ml , 1.0 mg / ml , 2.0 mg / ml , 3.0 mg / ml

The subcutaneous injectable solution was used as a medicament containing PEGylated recombinant granulocyte colony-stimulating factor (PEG-G-CSF) as an active ingredient and additional pharmacologically acceptable ingredients in the following proportions:

Active ingredient: PEGylated recombinant granulocyte colony-stimulating factor (PEG-G-CSF), 0.6-3.0 mg

Excipients:

Polysorbate 20 - 0.04 mg

Mannitol - 50 mg

Sodium acetic acid trihydrate - 0.23 mg

Glacial acetic acid pH 4.0 or less

Water for injection - up to 1ml.

Example  18

Example  3 PEG -G- CSF Of a final product such as an aqueous solution containing

Packaged in a syringe made of a hydrolysable class I neutral glass with a solder needle, capped with a syringe plunger, covered with a protective cap,

Packed in a bayer made of a butyl rubber cap with an aluminum cap or a hydrolyzable class I neutral glass clogged with Teflon-coated fluoro rubber.

Example  19

As active ingredients, Example  According to 3 The obtained PEG -G- CSF &Lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt;

A set containing one syringe complete with a piston in a contour cellular package made of a polymer film with instructions for use in a board package; or

A set containing one Bayer of contour blisters made of polymer film, packaged in a board package, with instructions for use.

Claims (17)

A conjugate of monomethoxy polyethylene glycol and human granulocyte colony stimulating factor, wherein the junction position of monomethoxy polyethylene glycol is N-terminal methionine of human granulocyte colony stimulating factor (G-CSF) and represented by the following formula (I)
(I)

In formula (I), n is an integer from 681 to 1000; m is an integer 4; N ^alpha HG-CSF is a natural or recombinant polypeptide having the activity of G-CSF,
Wherein said conjugate has at least 84% activity of unmodified G-CSF. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt; The conjugate according to claim 1, wherein the polyethylene glycol has an average molecular weight of 30 to 40 kDa. The conjugate according to claim 2, wherein the molecular weight of the polyethylene glycol is 30 kDa. Comprising an effective amount of a conjugate and a pharmaceutically acceptable excipient, wherein the conjugate comprises
A conjugate of monomethoxy polyethylene glycol and human granulocyte colony stimulating factor, wherein the junction position of monomethoxy polyethylene glycol is N-terminal methionine of human granulocyte colony stimulating factor (G-CSF) and represented by the following formula (I)
(I)

In formula (I), n is an integer from 681 to 1000; m is an integer 4; N ^alpha HG-CSF is a natural or recombinant polypeptide having the activity of G-CSF,
Also provided is a pharmaceutical composition for the treatment of neutropenia of various origins, characterized in that the conjugate has at least 84% activity of unmodified G-CSF. 5. The pharmaceutical composition according to claim 4, comprising polysorbate, mannitol, sodium acetate trihydrate, acetic acid and up to 1 ml of water for injection together with said effective amount of the conjugate. The pharmaceutical composition according to claim 5, wherein the pH value is 3 to 5. 7. The pharmaceutical composition according to claim 6, wherein the pH value is 4. A medicinal agent for the treatment of neutropenia of various origins, comprising a conjugate of formula (I) according to claim 1. A container sealed in a sterile environment, containing the pharmaceutical composition according to any one of claims 4 to 7. 10. The container of claim 9, wherein the container is a pre-filled syringe, bayer or autoinjector. A kit comprising the container according to claim 10 and prescription information. delete delete delete delete delete delete

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